Promiscuous G-protein coupled receptor inhibition of transient receptor potential melastatin 3 ion channels by Gβγ subunits

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Abstract

Transient receptor potential melastatin 3 (TRPM3) is a non-selective cation channel that is inhibited by Gβγ subunits liberated following activation of Gαi/o protein-coupled receptors. Here, we demonstrate that TRPM3 channels are also inhibited by Gβγ released from Gαs and Gαq. Activation of the Gs-coupled adenosine 2B receptor and the Gq-coupled muscarinic acetylcholine M1 receptor inhibited the activity of heterologously expressed TRPM3 in HEK293 cells. This inhibition was prevented when the Gβγ sink βARK1-ct (C-terminus of β-adrenergic receptor kinase-1) was co-expressed with TRPM3. In neurons isolated from mouse dorsal root ganglia (DRG), native TRPM3 channels were inhibited by activating Gs-coupled prostaglandin-EP2 and Gq-coupled bradykinin B2 (BK2) receptors. The Gi/o inhibitor pertussis toxin and inhibitors of PKA and PKC had no effect on EP2- and BK2-mediated inhibition of TRPM3, demonstrating that the receptors did not act through Gαi/o, or through the major protein kinases activated downstream of GPCR activation. When DRG neurons were dialysed with GRK2i, which sequesters free Gβγ protein, TRPM3 inhibition by EP2 and BK2 was significantly reduced. Intraplantar injections of EP2 or BK2 agonists inhibited both the nocifensive response evoked by TRPM3 agonists, and the heat-hypersensitivity produced by Freund's Complete Adjuvant (FCA). Furthermore, FCA-induced heat-hypersensitivity was completely reversed by the selective TRPM3 antagonist ononetin in wildtype mice and did not develop in Trpm3-/- mice. Our results demonstrate that TRPM3 is subject to promiscuous inhibition by Gβγ protein in heterologous expression systems, primary neurons and in vivo, and suggest a critical role for this ion channel in inflammatory heat hypersensitivity.

SIGNIFICANCE STATEMENT

The ion channel TRPM3 is widely expressed in the nervous system. Recent studies showed that Gαi/o-coupled GPCRs inhibit TRPM3 through a direct interaction between Gβγ subunits and TRPM3. Since Gβγ proteins can be liberated from other Gα subunits than Gαi/o, we examined whether activation of Gs- and Gq-coupled receptors also influence TRPM3 via Gβγ. Our results demonstrate that activation of Gs- and Gq-coupled GPCRs in recombinant cells and native sensory neurons inhibits TRPM3 via Gβγ liberation. We also demonstrated that Gs- and Gq-coupled receptors inhibit TRPM3 in vivo, thereby reducing pain produced by activation of TRPM3, and inflammatory heat hypersensitivity. Our results identify Gβγ inhibition of TRPM3 as an effector mechanism shared by the major Gα subunits.
Original languageEnglish
Pages (from-to)7840-7852
Number of pages13
JournalJournal of neuroscience
Volume39
Issue number40
Early online date26 Aug 2019
DOIs
Publication statusPublished - 2 Oct 2019

Keywords

  • DRG
  • G-protein beta-gamma
  • GPCR
  • ion channels
  • pain
  • TRPM3

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